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Hepatology. 2019 Nov 12. doi: 10.1002/hep.31037. [Epub ahead of print]

Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells.

Author information

1
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
2
Instituto de Investigación Sanitaria Aragón (IIS Aragón), Zaragoza, Spain.
3
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, The Netherlands.
4
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands.
5
Department of In Vitro Toxicology and Dermato-cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium.
6
Division of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.
7
Tytgat Institute for Liver and Intestinal Research, Gastroenterology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
8
Surgical Laboratory, Department of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
9
Cancer Genomics Netherlands, University Medical Center Utrecht, Utrecht, The Netherlands.
10
Princess Máxima Center, Utrecht, The Netherlands.
11
Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd), Madrid, Spain.
12
Fundación ARAID, Zaragoza, Spain.
13
Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.
14
Department of Biomedical and Aerospace Engineering, Universidad Carlos III de Madrid, Madrid, Spain.

Abstract

The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Here, we established a new method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after two weeks, compared to 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly upregulated compared to static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSION: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

KEYWORDS:

Liver organoids; hepatocyte differentiation; spinner flask; suspension culture; tissue engineering

PMID:
31715015
DOI:
10.1002/hep.31037

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